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You can clone with the same RE site on both ends of your construct, but it has several disadvantages. First, you have no control over the orientation of the insert -- it can go in forwards or backwards. Second, since you cut the vector only once, the vector can easily (and favorably!) religate rather than ligating your insert. This can sometimes be controlled by phosphate treatment of the vector after cutting, but this is (in my opinion) inferior in every way to using distinct enzymes at each end of your insert. The two enzymes must leave incompatible overhangs (that is, they cannot be isoschizimers). I'd recommend choosing enzymes that can be heat killed, avoiding a cleanup after RE digestion. The two RE digestions can be performed simultaneously in the same reaction in most cases (and you should choose enzymes that allow this). Most NEB enzymes now cut in buffer 4, for example.
#136265 Sequential restriction digestion of plasmid
Posted
on 22 June 2012 - 03:51 AM
